Bacterial live vector vaccines represent a vaccine development strategy that offers exceptional flexibility. With this approach, genes that encode protective antigens of unrelated bacterial, viral or parasitic pathogens are expressed in an attenuated bacterial vaccine strain that delivers these foreign antigens to the immune system, thereby eliciting a relevant immune response.
With the advent of powerful recombinant bioengineering techniques, it is now possible to genetically attenuate pathogenic bacteria to create safe and immunogenic live oral vaccines. Bacterial live vectors include attenuated enteric pathogens (e.g., Salmonella enterica, Shigella, Vibrio cholerae)2, 20, 21, 64, 84, 89, commensals (e.g., Lactobacillus, Streptococcus gordonii)62, 113 and licensed vaccine strains (e.g., BCG)29.
Such vaccines can be additionally engineered to express protective antigens from unrelated human pathogens, creating multivalent live vector vaccine strains. Typically, these foreign proteins are expressed within live vectors from multicopy expression plasmids that do not encode transfer functions and are not considered to be self-transmissible. Two fundamental lessons are becoming clear in live vector vaccinology: 1) multicopy expression plasmids can provide a gene dosage effect to enhance the level of expression of foreign antigens, and 2) in order to achieve enhanced immunogenicity from a gene dosage effect in live vectors, these multicopy plasmids must be genetically stabilized, particularly if expression of the foreign antigens metabolically stresses the live vector.
Antibiotic resistance markers are usually inserted into expression plasmids for selection purposes after introduction of plasmids into live vectors. Until recently, these resistance markers were considered to pose no risk for complicating or causing failure of clinical antimicrobial treatments for three important reasons: 1) the expression plasmids (and accompanying resistance markers) could not be efficiently mobilized from live vector donors to a recipient52, 2) the plasmid markers used encoded resistance to antibiotics not in widespread medical use, and 3) with no relevant antibiotic selective pressure, even rare plasmid transfers would not lead to de novo resistance becoming established within a new bacterial population52.
However, a growing body of evidence now clearly points to an inherent plasticity in the bacterial genome of intestinal microbes that allows rapid adaptation to environmental pressures using a striking variety of genetic mechanisms13, 43, 86. Indeed, intestinal bacteria have been proposed to act as a reservoir for mobile resistance cassettes and associated genes of metabolic importance, which cannot only be exchanged and maintained between resident flora of intestinal biofilms55, but might also be acquired or horizontally transferred to various genera of bacteria passing through the colon87. Examples of unexpected gene mobilization have recently been documented that challenge conventional thinking in bacterial genetics.
In elegant experiments designed to examine plasmid dynamics in biofilms, Maeda et al.58, 59 demonstrated the rapid transfer of a common multicopy pUC-like plasmid from a laboratory Escherichia coli K-12 DH5α strain to a recipient E. coli strain in the absence of antibiotic selection or any known fertility factors, R-factors, or other recognized conjugation or transduction functions. It was hypothesized that in situ horizontal transfer of plasmids occurred as DNA was released from dead and lysing “donor” bacteria and transferred into recipient bacteria by an unknown mechanism. Another unexpected example of in situ horizontal transfer was described by Ferguson et al.31, where conjugative plasmids were observed to be mobilized intracellularly at high frequency between Salmonella enterica strains residing within epithelial cell membrane-bound vacuoles. The frequency of plasmid transfer by conjugation was shown to be dependent on the probability of coinfection of the same epithelial cell by both donor and recipient; intracellular recombinants appeared by three hours after donor invasion and accumulated steadily over time. The authors posed the intriguing possibility of horizontal gene transfer between unrelated species of intracellular bacteria residing in the same target cell. Such examples clearly reveal the unexpected mobility of plasmids within a bacterial community, even in the absence of recognized selective pressures.
Genes encoding resistance to kanamycin (and the closely related antibiotic neomycin) have become the markers of choice for selection of recombinant plasmid DNA intended for use in human vaccines. These antimicrobials are used only occasionally in treatments of the gastrointestinal tract prior to elective colon surgery to avoid post-operative infection54, 88. Therefore, lack of routine clinical use of these antibiotics argues against selection and propagation of recombinant plasmids amongst intestinal bacteria. However, such reasoning does not hold up when applied to other bacterial ecosystems where sustained drug selection of resistance markers is not expected, such as amongst soil-borne microorganisms. A surprising diversity of stable resistance genes has now been documented in soil-dwelling bacteria with no obvious environmental exposure to antimicrobials25. It has been suggested that plasmid maintenance functions accompanying resistance genes provide a mechanism for persistence of these and other unrelated genes in the absence of selection91. Indeed, such maintenance systems have been intentionally engineered into expression plasmids carried by live vectors to enhance plasmid stability in vivo in the absence of drug selection37. Given the inherent unpredictability of plasmid mobilization between enteric strains, and the possibility of stable propagation in the absence of selection, the prospect of unintended and unforeseen genetic events compromising critical antimicrobial therapies cannot be formally excluded. Such risk is therefore unacceptable if alternatives to antibiotic selection can be developed.
Thus, there exists a need for non-antibiotic selection systems for live vector expression plasmids.